Tuberculosis is the leading cause of death due to a single infectious agent among adults in the world. The HIV-AIDS pandemic has had a strong influence on the incidence of tuberculosis in the developing world, greatly increasing the annual risk of developing tuberculosis in HIV positive individuals and contributing to the spread of tuberculosis in the community. The current tuberculosis vaccine, bacillus Calmette-Guerin (BCG) has a variable protective efficacy and can cause serious, even fatal disease in immunocompromised patients. An effective, inexpensive, easily distributed and administered subunit vaccine is required for the control of tuberculosis. The goal of this project is to investigate the ability of plant-derived, tuberculosis antigens to induce immune responses appropriate for the protection of mice against tuberculosis. The success of this project will be the first step in our overall goal of developing an inexpensive, subunit vaccine that will enable control of tuberculosis (TB) in humans including immunocompromised individuals. Investigations will include the ability of plants to correctly process and express protective antigens against Mycobacterium tuberculosis; verification of the mucosal immunogenicity of the resulting plant-derived antigens; characterization of the induced immune response(s) in mice and guinea pigs and the ability of the plant-derived antigens to protect animals from challenge with M. tuberculosis. Synthetic, plant optimized coding regions have been constructed for the tuberculosis antigens Ag85B and ESAT-6. These synthetic coding regions have been fused to the B and A2 subunits of the heat labile enterotoxin (LT) of enterotoxigenic Escherichia coil (ETEC) to promote targeting to mucosal lymphoid tissues. The resulting fused coding sequences were cloned into plant expression vectors for future Agrobacterium-mediated transformation of tomato (Lycopersicon esculentum L. cv "TA234). Expression, correct folding and concentration of the antigens in plant materials will be verified through Western analysis and enzyme linked immunosorbent assays (ELISA). Plant lines displaying high antigen expression, or elite plants, will be cloned, and transferred to the TB Research Materials and Vaccine Testing Contract at Colorado State University to be tested for mucosal immunogenicity and protective ability in mice trials.